Lactic acid bacterium having IgA production promoting activity, and use thereof

ABSTRACT

Disclosed are: a lactic acid bacterium belonging to Lactobacillus kunkeei, the bacterium having a higher IgA production inducing activity than that of Lactobacillus strain GG (ATCC53103), and a lower mitogenic activity and a lower IL-2 production inducing activity than those of Listeria strain EGD; and a food composition, a pharmaceutical composition, a cosmetic composition, an immunostimulant for preventing the infection by pathogens or viruses that invade through the respiratory or esophageal mucosa, and an intestinal immunostimulant for preventing or alleviating food poisoning, each of which contains the lactic acid bacterium or treated cells of the lactic acid bacterium.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of, and claims thebenefit of priority to, U.S. patent application Ser. No. 14/369,625,filed on Jun. 27, 2014, now U.S. Pat. No. 9,856,451, which is a NationalStage application under 35 U.S.C. § 371 of International Application No.PCT/JP2012/083523, having an International Filing Date of Dec. 25, 2012,which claims the benefit of priority of Japanese Application Serial No.2011-289314, having a filing date of Dec. 28, 2011. The disclosure ofeach of the foregoing applications is incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to a lactic acid bacterium belonging toLactobacillus kunkeei, which has a high IgA production inducing activityand is immunologically safe; and a food composition, a pharmaceuticalcomposition, a cosmetic composition, and an immunostimulant, each ofwhich comprises the lactic acid bacterium or treated cells of the lacticacid bacterium.

BACKGROUND ART

Among inflammatory bowel diseases (IBD), ulcerative colitis and Crohn'sdisease are of unknown cause, and are intractable. Factors associatedwith inflammatory bowel diseases are abnormalities in the intestinalbacterial flora, secretory IgA production disorder of intestinalepithelial cells, cytokines in the intestinal mucosa, and like factors.The primary lesion of ulcerative colitis is nonspecific chronicinflammation in the large intestine mucosa. Because variousautoantibodies are observed therein, ulcerative colitis is considered tohave characteristics as an autoimmune disease. On the other hand,Crohn's disease is pathologically characterized by noncaseatinggranulomatous inflammatory lesions, and dysfunction ofmonocyte/macrophage cells is considered to be one of the causativefactors for the disease. In Japan, the number of ulcerative colitispatients and Crohn's disease patients has steadily increased by slightlyless than 10 percent per year, which is problematic.

IBD used to be considered an autoimmune disease. However, with recentadvances in research, the inflammation has now been deemed to be causedby intestinal bacteria. It has been reported that intestinal mucosa ofIBD patients is less protective against endogenous microflora than incontrols, which results in an increase of bacteria in the lumen (seeGastroenterology, 1999, 117: 1089-1097). The use of probiotics as atherapy has been the focus of attention since probiotics were reportedto have an effect of preventing and alleviating enteritis. There havebeen many reports on the treatment of IBD with probiotics (Non-patentLiterature (NPL) 1).

Lactic acid bacteria, ingested in the form of fermented milk or thelike, are known to exhibit various physiological effects based on theirfunctionality, such as regulation of intestinal functions and reductionof serum cholesterol. As physiological effects of lactic acid bacteria,effects on autoimmune diseases such as chronic rheumatoid arthritis andinsulin-dependent diabetes, and inflammatory bowel diseases such asirritable bowel syndrome, ulcerative colitis, and Crohn's disease, havebegun to receive attention.

Patent Literature (PTL) 1 discloses that cell debris obtained bycrushing bacterial cells of one or more species of lactic acid bacteriaselected from the group consisting of lactic acid bacteria belonging tothe genera Lactobacillus, Lactococcus, and Streptococcus, hasinterleukin-10 and interleukin-12 production regulating ability, and theapplication of this product to Crohn's disease and refractoryinflammatory bowel diseases is disclosed.

Patent Literature (PTL) 2 discloses that lactic acid bacteria belongingto Lactobacillus salivarius, which are highly adhesive to mucousmembranes and have high proliferation ability and high resistance toacids, are applicable to prevention and/or treatment of ulcerativecolitis and Crohn's disease.

Non-patent Literature (NPL) 2 presents experimental results which showthat Lactobacillus casei strain GG has the potential to increase the gutIgA immune response and thereby to promote the gut immunological bather,and reports that Lactobacillus GG could provide an adjunct nutritionaltherapy for Crohn's disease.

Non-patent Literature (NPL) 3 reports research results which show thatLactobacillus GG may improve gut barrier function and clinical status inchildren with Crohn's disease.

Non-patent Literature (NPL) 4 reports that intranasal administration ofLactobacillus rhamnosus GG protects mice from influenza virus infectionby enhancing respiratory cell-mediated immune responses.

There is also a report that IFN-γ and IL-2 mRNA levels in the mucosa ofpatients with Crohn's disease are significantly increased, compared tocontrols, and that chronic intestinal inflammation in patients withCrohn's disease is characterized by an increase of Th1-like cytokines(Non-patent Literature (NPL) 5).

Non-patent Literature (NPL) 6 reports that Lactobacillus kunkeei, whichis a new species belonging to the genus Lactobacillus, was isolated fromwine. Further, Non-patent Literature (NPL) 7 reports that a fructophiliclactic acid bacteria strain was isolated from a fructose-rich site(flowers), and that the strain was found to contain Lactobacilluskunkeei. Patent Literature (PTL) 3 reports that Lactobacillus kunkeeiwas isolated from honeybees.

CITATION LIST Patent Literature

-   PLT 1: JP2007-269737A-   PLT 2: WO2002/016554-   PTL 3: JP2010-525809A

Non-Patent Literature

-   NPL 1: Journal of Intestinal Microbiology, 2009, 23: 193-201-   NPL 2: Annals of Nutrition and Metabolism, 1996, 40: 137-145-   NPL 3: Journal of Pediatric Gastroenterology and Nutrition, 2000,    31: 453-457-   NPL 4: Letters in Applied Microbiology, 2010, 50: 597-602-   NPL 5: Clinical and Experimental Immunology, 1995, 101: 428-435-   NPL 6: Journal of Applied Microbiology, 1998, 84: 698-702-   NPL 7: Systematic and Applied Microbiology, 2009, 32: 593-600

SUMMARY OF INVENTION Technical Problem

Lactic acid bacteria that have such excellent IgA production inducingactivity and that do not induce non-specific cell-mediated immuneresponses are highly useful, and the development of such lactic acidbacteria has been desired.

Accordingly, an object of the present invention is to provide a lacticacid bacterium belonging to Lactobacillus kunkeei, the bacterium havinghigh IgA production inducing activity and being immunologically safe.Another object of the present invention is to provide a foodcomposition, a pharmaceutical composition, a cosmetic composition, andan immunostimulant, each of which comprises the lactic acid bacterium ortreated cells of the lactic acid bacterium.

Solution to Problem

The present inventors conducted extensive research to achieve the aboveobjects. As a result, the inventors found that lactic acid bacteriabelonging to Lactobacillus kunkeei, which were isolated from bee pollenand identified, have a significantly higher IgA production inducingactivity than that of Lactobacillus strain GG (ATCC534103), and have asignificantly lower mitogenic activity and a significantly lower IL-2production inducing activity than those of Listeria strain EGD. Themitogenic activity and IL-2 production inducing activity of the lacticacid bacteria are substantially at the same levels as those achievedwithout stimulation, and are extremely low, even when compared withheat-killed Listeria bacteria. Therefore, these lactic acid bacteria areconsidered to have no ability to induce non-specific cellular immunity.That is, the lactic acid bacteria are immunologically safe materials.

The present invention was accomplished as a result of further researchbased on these findings, and provides the following lactic acidbacterium, compositions comprising the lactic acid bacterium, or thelike.

(I) Lactic Acid Bacterium

(I-1) A lactic acid bacterium belonging to Lactobacillus kunkeei, thebacterium having a higher IgA production inducing activity than that ofLactobacillus strain GG (ATCC53103), and a lower mitogenic activity anda lower IL-2 production inducing activity than those of Listeria strainEGD.(I-2) The lactic acid bacterium according to item (I-1), whichassimilates glucose, fructose, sucrose, trehalose, and gluconate.(I-3) The lactic acid bacterium according to item (I-1) or (I-2),wherein the source from which the lactic bacterium is isolated ishoneybees or apiculture products.(I-4) The lactic acid bacterium according to any one of items (I-1) to(I-3), which is Lactobacillus kunkeei BPS402 (FERM BP-11439) orLactobacillus kunkeei BPS104 (FERM BP-11438).(II) Composition 1(II-1) A food composition comprising the lactic acid bacterium accordingto any one of items (I-1) to (I-4), or treated cells thereof.(II-2) A pharmaceutical composition comprising the lactic acid bacteriumaccording to any one of items (I-1) to (I-4), or treated cells thereof.(II-3) A cosmetic composition comprising the lactic acid bacteriumaccording to any one of items (I-1) to (I-4), or treated cells thereof.(III) Immunostimulant(III-1) An immunostimulant for preventing infection by pathogens orviruses that invade through the respiratory or esophageal mucosa,comprising the lactic acid bacterium according to any one of items (I-1)to (I-4), or treated cells thereof.(III-2) An intestinal immunostimulant for preventing or alleviating foodpoisoning, comprising the lactic acid bacterium according to any one ofitems (I-1) to (I-4), or treated cells thereof.(IV) Composition 2(IV-1) A composition for intestinal regulation, beautification, oranti-aging, comprising the lactic acid bacterium according to any one ofitems (I-1) to (I-4), or treated cells thereof.(IV-2) A composition for preventing and/or treating an inflammatorybowel disease, comprising the lactic acid bacterium according to any oneof items (I-1) to (I-4), or treated cells thereof.(IV-3) The composition according to items (IV-2), wherein theinflammatory bowel disease is Crohn's disease or ulcerative colitis.(IV-4) The composition according to any one of (IV-1) to (IV-3), whichis a food or a medicament.

Advantageous Effect of the Invention

The lactic acid bacterium belonging to Lactobacillus kunkeei accordingto the present invention has excellent characteristics of having highIgA production inducing activity and being immunologically safe. Thelactic acid bacterium is useful in the fields of foods, drugs,cosmetics, etc.

Because the lactic acid bacterium of the present invention has the abovecharacteristics, the following effects are expected: immunostimulationfor preventing infection by pathogens or viruses that invade through therespiratory or esophageal mucosa; intestinal immunostimulation forpreventing or alleviating food poisoning; intestinal regulation,beautification, or anti-aging; and prevention and/or treatment of aninflammatory bowel disease, such as Crohn's disease or ulcerativecolitis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of agarose gel electrophoresis of RAPD PCR ofLactobacillus kunkeei strains BPS402 and BPS104, and strain JCM 16173,which were electrophoresed on 1% agarose gel at 50V for 1 hour and 30minutes and stained with EtBr.

FIG. 2 is a graph showing the IgA production inducing ability ofLactobacillus kunkeei strains BPS402 and BPS104. *:p<0.05 (vs. L.rhamnosus GG: ATCC53103, Dunnett's Multiple Comparison Test).

FIG. 3 is a graph showing mitogenic activity (S.I. value) ofLactobacillus kunkeei strains BPS402 and BPS104. *: p<0.01 (vs. Listeriamonocytogenes: EGD, Dunnett's Multiple Comparison Test), S.I. value:stimulation index, the amount of BrdU-Thymidine uptake by spleen-derivedlymphocytes (expressed with the uptake amount without stimulation beingset as 1.0).

FIG. 4 is a graph showing IL-2 production inducing ability (S.I. value)of Lactobacillus kunkeei strains BPS402 and BPS104. *: p<0.01 (vs.Listeria monocytogenes: EGD, Dunnett's Multiple Comparison Test), S.I.value: stimulation index, the amount of BrdU-Thymidine uptake by CTLL-2cells (expressed with the uptake amount without stimulation being set as1.0).

FIG. 5 is a graph showing a comparison of changes in the survival rateof BALB/c mice after infection with an influenza virus (A/PR/8/34strain) (a physiological saline-administered group vs. a 1mg/kg-administered group)

(**: significant differences at P<0.05 in comparison with thephysiological saline-administered group).

FIG. 6 is a graph showing a comparison of intrapulmonary viral genomeamounts in physiological saline-administered mice andBPS402-administered mice after infection with an influenza virus. InFIG. 6, the relative value of the amount of intrapulmonary virus genomein each sample is presented with standard deviations (**: significantdifferences at P<0.05).

FIG. 7 is a graph showing a comparison of virus-specific IgA inbronchoalveolar lavage fluid (BALF) between physiologicalsaline-administered mice and BPS402-administered mice after infectionwith an influenza virus. In FIG. 7, the average of the measurements ineach sample is presented with standard deviations (**: significantdifferences at P<0.05).

DESCRIPTION OF EMBODIMENTS

The present invention is described in detail below.

Lactic Acid Bacterium

The lactic acid bacterium belonging to Lactobacillus kunkeei accordingto the present invention is characterized by having a higher IgAproduction inducing activity than that of the Lactobacillus strain GG(ATCC534103), and a lower mitogenic activity and a lower IL-2 productioninducing activity than those of Listeria strain EGD.

The lactic acid bacterium of the present invention is preferably alactic acid bacterium that assimilates glucose, fructose, sucrose,trehalose, and gluconate. The source from which the lactic acidbacterium of the present invention is isolated is preferably honeybeesor apiculture products (honeybee products). In the present invention, anapiculture product refers to a product obtained by breeding honeybees.Examples of such products include honey, royal jelly, propolis, beeswax,bee bread, bee pollen, bee larvae, and processed products thereof (suchas extracts). A lactic acid bacterium isolated from bee pollen isparticularly preferable as the lactic acid bacterium of the presentinvention.

There are many reports on excellent IgA production inducing abilityprovided by oral administration of Lactobacillus strain GG (ATCC53103)to patients with Crohn's disease, etc. (Annals of Nutrition andMetabolism, 1996, 40: 137-145; Pediatric Research, 1992, 32: 141-144;Journal of Pediatric Gastroenterology and Nutrition, 1995, 20: 333-338;and FEMS Immunology and Medical Microbiology, 2000, 29: 47-52).Accordingly, a material having IgA production inducing activity higherthan that of Lactobacillus strain GG is considered to have remarkablyhigh IgA production inducing activity.

Further, Listeria (Listeria monocytogenes) is a gram-positive shortbacillus that causes listeriosis. Only viable bacteria thereof caninduce protective cellular immunity; establishing immunity withheat-killed bacteria is not easy (Japanese Bacteriology Magazine, 64(4):365-376, 2009).

Heat-killed Listeria strain EGD also has mitogenic activity andIL-2-inducing ability, although the levels of activity and ability arenot as high as in viable bacteria (Immunology, 1987, 62: 241-248).However, heat sterilization is actually effective for Listeria as wellas for many food-poisoning bacteria, and is widely known as a means forpreventing food poisoning. Accordingly, a material that has mitogenicactivity and IL-2 production inducing activity lower than those ofheat-killed Listeria strain EGD is considered to be immunologicallysafe.

Specific examples of the lactic acid bacterium belonging toLactobacillus kunkeei of the present invention include Lactobacilluskunkeei strain BPS402 and Lactobacillus kunkeei strain BPS104 isolatedfrom bee pollen and identified by the present inventors (hereinaftersometimes simply referred to as “strain BSP402” and “strain BSP104”).These strains were deposited with the National Institute of AdvancedIndustrial Science and Technology (Independent AdministrationCorporation), International Patent Organism Depositary (Chuo Dai-6, 1-1Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, (postal code:305-5466)) under the accession numbers FERM P-22177 and FERM P-22176 onOct. 3, 2011. These strains were transferred to an internationaldeposit, and have been deposited under the accession numbers FERMBP-11439 and FERM BP-11438.

The following are mycological properties and genetic properties of thestrains BPS402 and BPS104.

Lactobacillus kunkeei Strain BPS402

Mycological Properties

A. Morphological Characteristics

Cell morphology: bacillus (0.7-0.8×1.5-2.0 μm)

Gram stain: positive

Colony color: cream

48 hours of culture on MRS agar plate medium (30° C.)

B. Saccharide Fermentability (Using API 50 CH) Determined after 48 Hoursof Culture (Positive: +, Negative: −)

0 Control − 1 Glycerol − 2 Erythritol − 3 D-Arabinose − 4 L-Arabinose −5 D-Ribose − 6 D-Xylose − 7 L-Xylose − 8 D-Adonitol − 9Methyl-β-D-xylopyranoside − 10 D-Galactose − 11 D-Glucose + 12D-Fructose + 13 D-Mannose − 14 L-Sorbose − 15 L-Rhamnose − 16 Dulcitol −17 Inositol − 18 D-mannitol ? 19 D-sorbitol − 20Methyl-α-D-mannopyranoside − 21 Methyl-α-D-glucopyranoside − 22N-acetylglucosamine − 23 Amygdalin − 24 Arbutin − 25 Esculin ferriccitrate − 26 Salicin − 27 D-Cellobiose − 28 D-Maltose − 29 D-Lactose −30 D-Melibiose − 31 D-Sucrose + 32 D-Trehalose + 33 Inulin − 34D-Melezitose − 35 D-Raffinose − 36 Starch − 37 Glycogen − 38 Xylitol −39 Gentiobiose − 40 D-Turanose − 41 D-Lyxose − 42 D-Tagatose − 43D-Fucose − 44 L-Fucose − 45 D-Arabitol − 46 L-Arabitol − 47 Gluconate +48 2-Keto-gluconate − 49 5-Keto-gluconate −Genetic Properties

SEQ ID NO: 1 in the sequence list shows the base sequence of 16S rRNA ofthe strain BPS402. The sequence of 16S rRNA of the strain BPS402 has100% identity to Lactobacillus kunkeei strain YH-15 (JCM16173), which isa reference strain of a known species. The results of simplifiedmolecular phylogenetic analysis showed that the strain BPS402 formed acluster together with Lactobacillus kunkeei, and that both were in thesame molecular phylogenetic position. It is determined from theseresults that the strain BPS402 is a lactic acid bacterium belonging toLactobacillus kunkeei.

Lactobacillus kunkeei Strain BPS104

Mycological Properties

A. Morphological Characteristics

Cell morphology: Bacillus (0.6-0.7×1.2-1.5 μm)

Gram stain: positive

Colony color: milky-white

72 hours of culture on an MRS agar plate medium (30° C.)

B. Saccharide Fermentability (Using API 50 CH) Determined after 48 Hoursof Culture (Positive: +, Negative: −)

0 Control − 1 Glycerol − 2 Erythritol − 3 D-Arabinose − 4 L-Arabinose −5 D-Ribose − 6 D-Xylose − 7 L-Xylose − 8 D-Adonitol − 9Methyl-β-D-xylopyranoside − 10 D-Galactose − 11 D-Glucose + 12D-Fructose + 13 D-Mannose − 14 L-Sorbose − 15 L-Rhamnose − 16 Dulcitol −17 Inositol − 18 D-Mannitol ? 19 D-Sorbitol − 20Methyl-α-D-mannopyranoside − 21 Methyl-α-D-glucopyranoside − 22N-Acetylglucosamine − 23 Amygdalin − 24 Arbutin − 25 Esculin FerricCitrate − 26 Salicin − 27 D-Cellobiose − 28 D-Maltose − 29 D-Lactose −30 D-Melibiose − 31 D-Sucrose + 32 D-Trehalose + 33 Inulin − 34D-Melezitose − 35 D-Raffinose − 36 Starch − 37 Glycogen − 38 Xylitol −39 Gentiobiose − 40 D-Turanose − 41 D-Lyxose − 42 D-Tagatose − 43D-Fucose − 44 L-Fucose − 45 D-Arabitol − 46 L-Arabitol − 47 Gluconate +48 2-Keto-gluconate − 49 5-Keto-gluconate −Genetic Properties

SEQ ID NO: 2 in the sequence list shows the base sequence of 16S rRNA ofthe strain BPS402. The sequence of 16S rRNA of the strain BPS104 has99.9% identity to Lactobacillus kunkeei strain YH-15 (JCM16173), whichis a reference strain of a known species. The results of simplifiedmolecular phylogenetic analysis showed that the strain BPS104 formed acluster together with Lactobacillus kunkeei, and that both were in thesame molecular phylogenetic position. It is determined from theseresults that the strain BPS104 is a lactic acid bacterium belonging toLactobacillus kunkeei.

Table 1 below shows the summary of test results of saccharidefermentability of the strain BPS402, strain BPS104, and Lactobacilluskunkeei (JCM16173). Table 1 shows that the saccharide fermentability ofthe strain BPS402 and strain BPS104 is different from that of the knownstrain.

TABLE 1 Glucose Fructose Sucrose Trehalose Gluconate L. kunkeei + − + −− (JCM16173) BPS402 + + + + + BPS104 + + + + + Results of determinationafter 48 hours Positive: +, Negative: −

Random amplified polymorphic DNA (RAPD) analysis was performed using aprimer 5′-CCGCAGCCAA-3′ under the following reaction conditions.Preincubation: 94° C. for 2 minutes, amplification: 94° C. for 1 minute,30° C. for 1 minute, and 72° C. for 1.5 minutes (40 cycles), and primerextension: 74° C. for 5 minutes (1 cycle).

FIG. 1 shows the results. The results of FIG. 1 show that the RAPDpatterns of the strains BPS402 and BPS104 are different from those ofknown Lactobacillus kunkeei strains.

It was determined from these results that the strains BPS402 and BPS104are novel Lactobacillus kunkeei strains.

These strains can be cultured in a usual manner. There is no specificlimitation on the medium to be used, as long as these strains can becultured therein. Natural media, synthetic media, semisynthetic media,and like media can be used. Milk, royal jelly, etc., can also be used asmedia. As the medium, media containing a nitrogen source and a carbonsource can be used. Examples of nitrogen sources include meat extract,peptone, casein, yeast extract, gluten, soybean flour, soybeanhydrolyzate, amino acids, and the like. Examples of carbon sourcesinclude glucose, lactose, fructose, inositol, sorbitol, starch syrup,starch, rice malt extract, wheat bran, bagasse, molasses, and the like.Further, minerals (such as ammonium sulfate, potassium phosphate,magnesium chloride, sodium chloride, calcium carbonate, iron, manganese,and molybdenum), various vitamins, etc. can be added.

The culturing temperature is typically 4 to 45° C., and preferably 30 to37° C. The culturing time is typically about 8 to 72 hours.Aeration-shaking or aeration-agitation may be performed. The pH of themedium is typically 4.0 to 9.0, and preferably 6.0 to 8.0.

An example of the culture method is a method comprising inoculating thecells at a concentration of 1% into MRS medium, and culturing the cellsat 37° C. for 24 hours.

Food Composition

The food composition of the present invention comprises the lactic acidbacterium or treated cells of the lactic acid bacterium as an essentialcomponent. The food composition includes all food compositions that canbe ingested by animals (including humans).

The food composition of the present invention contains the lactic acidbacterium that may be either viable or heat-killed. As the lactic acidbacterium, isolated cells may be used, or a cell culture or fermentationproduct thereof may be used.

Examples of the treated cells of the lactic acid bacterium include thoseobtained by subjecting the lactic acid bacterium to heating, pasting,drying (freeze-king, vacuum-drying, spray-king, etc.), freezing, lysis,crushing, extraction, etc. Examples of the treated cells further includesupernatants obtained by removing proteins from cell debris obtained bysonication or other disintegration techniques, supernatants obtained byremoving the solids from a cell culture or fermentation product thereof,etc.

The lactic acid bacterium may be used alone, or in admixture with othermicroorganisms.

To prepare the food composition of the present invention, the lacticacid bacterium or treated cells thereof may be used without furtherprocessing, or, if necessary, may be mixed with minerals, vitamins,flavonoids, quinones, polyphenols, amino acids, nucleic acids, essentialfatty acids, fresheners, binders, sweeteners, disintegrators,lubricants, coloring agents, fragrances, stabilizing agents,preservatives, sustained-release regulators, surfactants, solubilizers,wetting agents, and the like.

The type of food composition according to the present invention is notparticularly limited, and examples thereof include dairy products;fermented foods (yogurt, royal jelly, etc.); beverages (coffee, juices,tea drinks, and like soft drinks, milk beverages, lactic acid bacteriabeverages, drinks containing lactic acid bacteria, yogurt drinks,carbonated drinks, Japanese sake, Western wines and spirits, fruitwines, and like liquors, etc.); spreads (custard cream, etc.); pastes(fruit pastes, etc.); Western-style confectioneries (chocolate,doughnut, pie, cream puff, gum, jelly, candies, cookies, cakes, pudding,etc.); Japanese sweets (daifuku [rice cake stuffed with sweet beans],rice cake, manju [steamed yeast bun with filling], kasutera [Castellasponge cakes], anmitsu [gelatin mixed with other ingredients], yokan[azuki bean jelly], etc.); frozen desserts (ice creams, ice candies,sherbets, etc.); foods (curry, gyudon [beef and onion stew on top of abowl of rice], zousui [risotto], miso soup, soup, meat sauce, pasta,pickles, jam, royal jelly, etc.); seasonings (dressing, furikake [amixture of dried fish and/or vegetable flakes for sprinkling on steamedrice], tasty seasonings, soup bases, etc.); etc.

The method for producing the food composition of the present inventionis also not particularly limited, and can be suitably performedaccording to a known method. For example, the lactic acid bacterium ortreated cells thereof may be mixed into or sprayed over the intermediateor final product obtained in a process of producing such a foodcomposition as described above, thereby obtaining a food. Fermentedproducts, lactic acid bacteria beverages, and drinks containing lacticacid bacteria can be obtained by fermenting cow's milk or like animalmilks, milk raw materials, etc., with the lactic acid bacterium. Thefermented foods of royal jelly can also be produced by fermenting royaljelly with the lactic acid bacterium.

The food composition of the present invention can also be used as healthfoods, functional foods, nutraceuticals, dietary supplements, foods forhealth uses, foods for specified health uses, or probiotic products. Thedosage unit form of the composition for use as a dietary supplement isnot particularly limited, and can be suitably selected. Examples of suchdosage forms include tablets, capsules, granules, liquids, powders, andthe like.

The content of the lactic acid bacterium or treated cells thereof in thefood composition of the present invention can be suitably selected from1×10⁻⁷ to 100 wt. %, preferably 1×10⁻⁵ to 100 wt. %, and more preferably1 to 100 wt. %.

The amount of intake of the food composition of the present inventioncan be suitably set according to various conditions, such as theconsumer's body weight, age, sex, and symptoms. The daily intakeexpressed by the number of lactic acid bacterium may be, for example,1×10⁶ or more, preferably 1×10⁹ or more, and more preferably 1×10¹² ormore.

Pharmaceutical Composition

The pharmaceutical composition of the present invention comprises thelactic acid bacterium or treated cells thereof as an essentialingredient. The pharmaceutical composition of the present invention canbe referred to as a lactic acid bacteria preparation.

The pharmaceutical composition of the present invention contains thelactic acid bacterium that may be either viable or heat-killed. As thelactic acid bacterium, isolated cells may be used, or a cell culture orfermentation product thereof may be used.

Examples of the treated cells of the lactic acid bacterium include thoseobtained by subjecting the lactic acid bacterium to heating, pasting,drying (freeze-drying, vacuum-drying, spray-drying, etc.), freezing,lysis, etc. Examples of the treated cells further include supernatantsobtained by removing proteins from cell debris obtained by sonication orother disintegration techniques, supernatants obtained by removing thesolids from a cell culture or fermentation product thereof, etc.

The lactic acid bacterium may be used alone, or in admixture with othermicroorganisms.

To prepare the pharmaceutical composition, the lactic acid bacterium ortreated cells thereof may be used without further processing, or mixedwith a pharmaceutically acceptable non-toxic carrier, diluent, orexcipient, and formed into tablets (including uncoated tablets,sugar-coated tablets, effervescent tablets, film-coated tablets,chewable tablets, troches, etc.), capsules, pills, powder (powdereddrugs), fine granules, granules, liquids, suspensions, emulsions,syrups, pastes, and injections (including preparations formulated intoliquids by admixture into distilled water or an infusion, such as anamino acid infusion or an electrolyte infusion, before use).

The content of the lactic acid bacterium or treated cells thereof in thepharmaceutical composition of the present invention can be suitablyselected from 1×10⁻⁷ to 100 wt. %, preferably 1×10⁻⁵ to 100 wt. %, andmore preferably 1 to 100 wt. %.

The dose of the pharmaceutical composition of the present invention canbe suitably set according to various conditions, such as the patient'sbody weight, age, sex, and symptoms. The daily dose expressed by thenumber of lactic acid bacterium may be, for example, 1×10¹⁰ or more,preferably 1×10¹¹ or more, and more preferably 1×10¹² or more.

The lactic acid bacterium or treated cells thereof according to thepresent invention can also be used as an immunostimulant for preventinginfection by a pathogen or a virus that invades through the respiratoryor esophageal mucosa, or as an intestinal immunostimulant for preventingor alleviating food poisoning. Examples of the virus that therespiratory or esophageal mucosa include, but are not limited to,influenza viruses.

The food or pharmaceutical composition of the present invention iseffective for intestinal regulation, beautification, or anti-aging, andpreventing and/or treating inflammatory bowel diseases (preferablyCrohn's disease or ulcerative colitis).

Cosmetic Composition

The cosmetic composition of the present invention contains the lacticacid bacterium or treated cells thereof as an essential component.

The cosmetic composition of the present invention contains the lacticacid bacterium that may be either viable or heat-killed. As the lacticacid bacterium, isolated cells may be used, or a cell culture orfermentation product thereof may be used.

Examples of the treated cells of the lactic acid bacterium include thoseobtained by subjecting the lactic acid bacterium to heating, pasting,drying (freeze-drying, vacuum-drying, spray-drying, etc.), freezing,lysis, etc. Examples of the treated cells further include supernatantsobtained by removing proteins from cell debris obtained by sonication orother disintegration techniques, supernatants obtained by removing thesolids from a cell culture or fermentation product thereof, etc.

The lactic acid bacterium may be used alone, or in admixture with othermicroorganisms.

The cosmetic composition of the present invention includes all cosmeticcompositions that are applied to the skin, mucosa, body hair, head hair,scalp, nails, teeth, facial skin, lips, etc. of animals (includinghumans).

The content of the lactic acid bacterium or treated cells thereof in thecosmetic composition of the present invention can be suitably selectedfrom the range of 1×10⁻¹⁰ to 100 wt. %, preferably 1×10⁻⁶ to 50 wt. %,and more preferably 1×10⁻² to 10 wt. %.

The cosmetic composition of the present invention may contain, inaddition to the lactic acid bacterium or treated cells thereof,ingredients commonly used in cosmetics, such as skin-lightening agents,moisturizers, antioxidants, oily components, ultraviolet absorbers,surfactants, thickeners, alcohols, powder constituents, color materials,aqueous ingredients, water, various skin nutrients, etc.

The cosmetic composition of the present invention can be formulated intoa wide range of dosage forms, such as aqueous solutions, solubilizers,emulsions, powders, oils, gels, ointments, aerosols, water-oil bilayerpreparations, and water-oil-powder trilayer preparations.

The cosmetic composition of the present invention may be used for anypurpose of use. Examples of such uses include basic cosmetics such asfacial washes, face lotions, milky lotions, creams, gels, essences,beauty essences, packs, and masks; make-up products such as foundations,lipsticks, blushes, eye shadows, eyeliners, and mascaras; nail cosmeticssuch as nail polishes, base coats, topcoats, and nail polish removers;and other products such as facial washes, massaging agents, cleansingagents, after-shave lotions, pre-shave lotions, shaving creams, bodysoaps, soaps, shampoos, rinses, hair treatments, hair dressings, hairtonics, hair growing agents, antiperspirants, and bath agents.

The cosmetic composition of the present invention is expected to exhibitanti-aging effects, such as skin-lightening effects and moisturizingeffects.

EXAMPLES

The present invention is described below in more detail with referenceto Examples. However, the present invention is not limited thereto orthereby.

Screening of Lactobacillus kunkeei Strains BPS402 and BPS104

The medium used for isolating the bacteria was an MRS culture medium (orROGOSA medium). Any of apple juice, orange juice, tomato juice, or grapejuice was added as desired to achieve a final concentration of 25%, andthe medium was adjusted to pH 4.5 with acetic acid and sodium hydroxide.Bee pollen was appropriately diluted in the prepared medium, and culturewas started at 25 to 37° C. in an incubator. After 2 to 3 days ofculture, the cells were spread on MRS agar medium containing calciumcarbonate by a streak method, and culture was further continued at thesame temperature as above for another two to three days to obtain singlecolonies having a clear zone by lactic acid production. The obtainedcolonies were cultured in MRS, and then cryopreserved at −80° C. as a10% glycerol stock.

In particular, the amount of IgA production (O.D.≥0.2), and mitogenicactivity and IL-2 production (S.I. value≤1.2) were used as screeningindices in the present test examples. For mitogenic activity and IL-2production, S.I. value without stimulation was set as 1.0.

TEST EXAMPLES Test Example 1

Experimental Method

Breeding/Feeding of Animals

Six-week-old female BALB/c mice preliminarily bred for one week aftercompletion of quarantine and inspection were used. The individuals thathad similar body weights and exhibited similar changes in body weightduring the preliminary breeding were selected as test animals.

Preparation of Cells

In accordance with the test animal breeding and feeding laws, the bloodwas collected from the animals under anesthesia to exsanguinate theanimals. After the exsanguination, spleen and small intestine (includingcecum and ileocecum) were excised, and transferred to Petri dishescontaining a 1% BSA-containing Hank's solution, and stored ice-cold.Spleen-derived lymphocytes (SPL) were collected from the spleen, andPeyer's patch-derived lymphocytes (PPL) were collected from the smallintestine. Cells of each type were resuspended in 10% RPMI-1640 mediumto a density of 1×10⁶ cells/ml.

Preparation of Bacteria

Each bacterial strain was streaked on Mueller-Hinton agar medium, andcultured in a 37° C. incubator for 16 hours. Single colonies grown onthe agar medium were picked up with a platinum needle, streaked on newMueller-Hinton agar medium, and cultured in a 37° C. incubator for 16hours. The cell colonies were lysed in TS liquid medium to a McFarlandturbidity (MCF) of 3.0 as a rule of thumb, thus preparing bacterialsuspensions. The number of cells in a portion of each bacterialsuspension was counted by the tenfold serial dilution method. Theobtained bacterial suspensions were heated at 100° C. for 30 minutes,and used as the test substances described in the next section. As acontrol bacterial strain, Listeria monocytogenes (EGD strain) was used,and the same procedure as the above streak culture was performed to anMCF of 3.0 as a rule of thumb to prepare a bacterial suspension.Further, the obtained bacterial suspension was heated at 100° C. for 30minutes, and used as the control strain described in the next sectionIgA production inducing ability measurement test

The collected PPL was stimulated with one of the test substances as anantigen. Forty-eight hours after the stimulation, the culturesupernatant was collected. IgA in the collected culture supernatant wasmeasured by ELISA assay.

Lymphocyte Blastogenesis Test

The collected SPL was stimulated with mitogen (PWM final concentration:5 μg/mL) or with one of the test substances as an antigen in thepresence of BrdU-Thymidine. The amount of uptake of BrdU-Thymidine wasmeasured. Further, the culture supernatant was collected using the samesystem, and a cell proliferation ability test using CTLL-2 cells wasconducted.

Results and Discussion

IgA Antibody Production Inducing Ability

Peyer's patch cells were stimulated with one of the test substances. Theculture supernatant after 48 hours of stimulation was added to an ELISAplate for measuring IgA antibody, and IgA antibody production inducingability was evaluated. Peyer's patch cells without stimulation were usedas a negative control, and the O.D. value obtained using each testsubstance was evaluated relative to the O.D. value of the negativecontrol. FIG. 2 shows the results.

The O.D. values of the strains BPS402 and BPS104 were 0.243 and 0.242,respectively, and the O.D. value obtained using the control strainListeria monocytogenes was 0.107.

Lymphocyte Blastogenesis Response

Spleen Cell Proliferation Responses Determined Using BrdU Uptake as anIndicator

Spleen cells were stimulated using one of the test substances. Theuptake of fluorescently labeled BrdU-Thymidine was evaluated byfluorescence intensity using a flow cytometer. Spleen cells withoutstimulation were used as a negative control. The fluorescence intensityobtained using each test substance was evaluated relative to thefluorescence intensity of the negative control. FIG. 3 shows theresults.

The S.I. value of the blastogenic response obtained using the controlstrain Listeria monocytogenes was 2.1, whereas S.I. values of thestrains BPS402 and BSP104 were 1.2 and 0.9, respectively.

Evaluation of Test Substances Using CTLL-2 Cell Proliferation Responseas an Indicator

IL-2 in the culture supernatant obtained in the above test was measuredusing CTLL-2 cells as an indicator. For the measurement, BrdU-Thymidineuptake of CTLL-2 cells was evaluated by fluorescence intensitydetermined using a flow cytometer. Spleen cells without stimulation wereused as a negative control. The fluorescence intensity of each testsubstance was evaluated relative to the fluorescence intensity of thenegative control. FIG. 4 shows the results.

The S.I. value of the blastogenic response obtained using the controlstrain Listeria monocytogenes was 1.4, while the S.I. values of thestrains BPS402 and BSP104 were 1.1 and 1.0, respectively.

The heat-killed lactic acid bacterial strains BPS402 and BPS104 did notexhibit such potent mitogenic activity as to induce T cell immuneresponse. Even compared to heat-killed Listeria bacteria, which areintracellular parasitic bacteria, the heat-killed lactic acid bacterialstrains BPS402 and BPS104 are extremely low in terms of cellularproliferation response and IL-2 production inducing ability, and thus donot have the ability to non-specifically induce cellular immunity. Thatis, the strains BPS402 and BPS104 do not always enhance immune activityin a host, and are considered to be immunologically safe substances.

It was suggested that the heat-killed lactic acid bacteria may induceIgA production of Peyer's patch cells. Accordingly, the heat-killedlactic acid bacteria are likely to increase the amount of IgA antibodypresent in mucosal epithelium, such as respiratory mucosa and esophagealmucosa, and is thus considered to be effective for preventing infectionof pathogens (such as viruses) that invade through the mouth and nose.

Test Example 2

Test Method

1. Preparation of Virus

MDCK cells cultured in a cell growth medium were infected with aninfluenza virus (IFVA, strain PR/8/34(H1N1)), which had beencryopreserved in a deep freezer, at a multiplicity of infection (M.O.I.)of 0.01 and cultured at 37° C. in the presence of 5% CO₂ for 72 hours(one passage). Cells serially passaged for 5 passages weremass-produced, and subjected to sucrose gradient centrifugation toseparate and purify a viral fluid. The viral fluid was stored in a deepfreezer until use. A portion of the virus fluid was subjected to tenfoldserial dilution to confirm cytopathic effects, thereby determining theviral infectivity titer (TCID₅₀) and plaque-forming unit (pfu).

2. Viability Analysis

a. Oral Administration of Test Substance

Six-week-old male BALB/c mice preliminarily bred for one week aftercompletion of the quarantine and inspection were used. Each mouse wasgiven one of the test substances in an amount of 0.2 mL by a single oralgavage. The concentration of each test substance was set to 1 mg per kgof the body weight. A test substance was given by oral gavage once a daythroughout the test period, and this administration was continued untilthe end of the test period after IFVA infection. Physiological salinewas orally administered to a negative control group in the same manneras each test substance.

b. Grouping

The mice were divided into two test groups. The table below shows thedetails. The two groups were intranasally inoculated with the preparedIFVA, and 20 mice reliably inoculated with the virus were assigned toeach group. For grouping, each mouse was numbered and assigned bystratified randomization when the test substance began to beadministered.

TABLE 2 Test substance Test group Viral infection concentration Numberof mice 1 + − 20 (Intranasal inoculation) 2 + 1 mg/kg 20c. IFVA Inoculation

Nine-week-old female BALB/c mice given the test substance by oral gavagefor 3 consecutive weeks were used. The mice were intraperitoneallyinjected with Pentobarbital sodium (5.0 mg/mL; KyoritsuseiyakuCorporation (trade name; Somnopentyl anesthetic injection) diluted withphysiological saline at 50 mg/kg in the order of animal numbering toundergo general anesthesia Under general anesthesia, the prepared IFVAwas intranasally inoculated at 103 pfu/20 μL into the right nasal cavityof each mouse.

d. Determination of the Survival Rate

To determine the survival rate, follow-up observations were performedfor 3 weeks after IFVA inoculation. The survival rate was determined bydividing the number of individuals that survived during the follow-upobservation period by the total number of individuals in each group, andmultiplying the obtained value by 100.

3. Immunological Analysis

a. Oral Administration of Test Substance

Six-week-old male BALB/c mice preliminarily bred for one week aftercompletion of the quarantine and inspection were used. Each mouse wasgiven the test substance in an amount of 0.2 mL by a single oral gavage.The concentration of the test substance was set to 100 mg per kg of thebody weight. The test substance was given by oral gavage once a daythroughout the test period, and this administration was continued untilthe end of the test period after IFVA infection. Physiological salinewas orally administered to a negative control group in the same manneras each test substance.

b. Grouping

The mice were divided into a total of four groups: two virus-inoculatedgroups, and two non-virus-inoculated groups. The table below shows thedetails. The two virus-inoculated groups were intranasally inoculatedwith the prepared IFVA, and 50 mice reliably inoculated with the viruswere assigned to each group. The number of mice in each of thenon-virus-inoculated groups was 10. For grouping, each mouse wasnumbered and assigned by stratified randomization when the testsubstance began to be administered.

TABLE 3 Test Viral Test substance Number group infection concentrationof mice Notes 1 − − 10 For analysis of (none) (Physiologicalnon-infection (d 0) saline) 2 − 100 mg/kg 10 For analysis ofnon-infection (d 0) 3 + − 50 (Intranasal inoculation) 4 + 100 mg/kg 50c. IFVA Inoculation

Nine-week-old female BALB/c mice given the test substance by oral gavagefor 3 consecutive weeks were used. Pentobarbital sodium (5.0 mg/mL;Kyoritsuseiyaku Corporation (trade name; Somnopentyl anestheticinjection) diluted with physiological saline was intraperitoneallyinjected to the mice at 50 mg/kg in the order of animal numbering toundergo general anesthesia Under general anesthesia, the prepared IFVAwas intranasally inoculated at 102 pfu/20 μL into the right nasal cavityof each mouse. 20 μL of physiological saline was intranasallyadministered to the non-virus-inoculated groups in a similar manner.

d. Collection of Tissue Samples

0 day (d0) and 14 days (d14) after the IFVA infection, eight mice ineach group were killed. Bronchioalveolar lavage fluid (BALF) wascollected from five mice in each group, while untreated lungs wereexcised from three mice in each group and preserved in 10% (v/v)formalin.

e. Measurement of the Amount of Pulmonary Viral Genomic RNA

The lungs after the treatment were excised from 5 mice in each group,and cell suspensions were created using a gentle MACS Dissociator(Miltenyi Biotec). Lung-derived total RNA was extracted from each of thecell suspensions using ISOGEN II. The extracted RNA was subjected to areverse transcription reaction using Uni12 primer (Hoffmann E. et al.,Arch Virol., 2001). After selectively amplifying viral genomic RNA, theamount of viral genomic RNA in the lungs was measured by real-time PCRusing primers specific to the viral NP gene.

f. Antibody Measurement

The collected BALF was centrifuged and separated into a supernatant andcellular components. The supernatant was stored in a deep freezer untilanalysis was conducted. The antibody in the BALF supernatant wasmeasured by ELISA. The measurement parameter was anti-influenzavirus-specific IgA.

4. Statistical Processing

A comparison in the survival rate analysis between the groups was madeusing a log-rank test at a two-sided significance level of 0.05.Significance of difference between the groups in other tests wasassessed using a Student's t-test at a two-sided significance level of0.05.

Results and Discussion

1. Survival Rate Analysis (FIG. 5)

The physiological saline-administered group, which is an experimentalcontrol group, had a survival rate of 25.0% at the completion of thetest. The group given a test substance (lactic acid bacteria BPS402powder; hereinafter referred to as BPS402) in an amount of 1 mg/kg had asurvival rate of 55.0% at the completion of the test. A comparison ofthe group given BPS402 in an amount of 1 mg/kg to the experimentalcontrol group using a log-rank test (p=0.0465) indicated a significantdifference.

The results show that the BPS402-administered group tends to have higherresistance to IFVA infection than the experimental control group.Non-patent Literature (NPL) 4 (Letters in Applied Microbiology, 2010,50: 597-602) discloses that administration of LGG in an amount of 200μg/mouse provides an influenza virus-preventing effect, whereas the useof BPS402 in an amount of 20 μg/mouse (the body weight of the mouse isassumed to be 20 g) provided the preventing effect. This indicates thatBPS402 was effective at one-tenth the dose of the LGG

2. Immunological Analysis

a. Measurement of the Amount of Pulmonary Viral Genomic RNA (FIG. 6)

A signification reduction in viral load in the BPS402-administered group14 days after the infection was observed.

b. Antibody Measurement (FIG. 7)

A significant increase in the amount of influenza virus-specific IgA wasobserved in the BALF of IFVA-infected, BPS402-administered mice 14 daysafter the infection.

As a result of in vivo immunological analysis using theBPS402-administered mice, an elevation of influenza virus-specificsecretory IgA in the lung alveoli and bronchi at the late infectionstage, and enhanced antiviral effects at the late infection stage wereobserved in the BPS402-administered group. The results suggest thatBPS402 may play a role in quickly overcoming the infection by exerting aparticularly strong effect on B-cell lineage plasma cells, and causingefficient secretion of influenza virus-specific IgA.

Test Example 3

The efficacy and safety of Lactobacillus kunkeei BPS402 in humans wereconfirmed in this test.

General paid volunteers who were healthy but tended to be constipatedwere subjected to a double-blind, placebo-controlled trial. The subjectswere paid volunteers aged 40 or older. These subjects were divided intotwo groups, seven subjects in a BPS402 group and seven subjects in aplacebo group, in such a manner that they were as equal as possible ineach item. The amount of intake was 5 doses (1,000 mg) per day, and theintake period was four weeks.

Efficacy

A significant elevation of sIgA (μg/min) in saliva was observed afterintake of BPS402, compared to that before intake of BPS402. sIgA iswidely known as an immunoglobulin having important immunologicalfunction in the mucosal system, and is considered to reduce the risk ofinfection with upper respiratory tract infections.

The degree of dryness of the skin was shown on a linear scale of 0 to100 mm and analyzed by visual analog scale (VAS). Dryness of the skinwas significantly improved after intake of BPS402, compared to thatbefore intake of BPS402, and a moisturizing effect was confirmed.

Safety

Intake of BPS402 created no particular problem in safety.

Production Examples

Production Examples of the compositions of the present invention areshown below.

Production Example of Lactic Acid Bacteria Powder

1,000 L of a medium containing 2% glucose and 5% yeast extract wasadjusted to pH 6.5 and BPS104 culture obtained by preculture wasinoculated thereinto at 1%. While the pH was suitably adjusted, thecells were cultured at 30 to 37° C. for 24 to 48 hours to obtain a cellculture. The resulting cell culture was centrifuged, and 1 part byweight of water was added per part by weight of the cells. Then, thecells were lyophilized, and the solids were pulverized by a pulverizerto obtain 1 kg of lactic acid bacteria BPS104 powder.

Production Example of Lactic Acid Bacteria Capsules

400 g of lactic acid bacteria BPS104 powder and 20 g of sucrose fattyacid ester were mixed. The mixture was filled into hard capsules using afiller to obtain 2,000 capsules of lactic acid bacteria BPS104.

Production Example of Lactic Acid Bacteria Fermented Extract

Example 1

After 1,000 mL of a diluent prepared by diluting royal jelly to a finalconcentration of 5% was adjusted to pH 6.5, BPS402 culture obtained bypreculture was inoculated at 1%. After culture at 30° C. for 48 hours,the residue was removed by centrifugation, and 800 mL of a royal jellyfermented extract was obtained.

Example 2

The same procedure as in Example 1 was repeated to obtain 800 mL ofenzyme-treated royal jelly from 1,000 mL of a diluent obtained bydiluting enzyme-treated royal jelly fermented extract to a finalconcentration of 5%.

The fermented extracts obtained in Examples 1 and 2 were tested in thefollowing five items. No effect was indicated as −, and a slight effectwas indicated as −/+. Effects were evaluated on a scale of one (+) tothree (+++), in ascending order of effects. Table 4 shows the results.

pH Reduction

The fermented extract after 48 hours was evaluated using a pH meter.

Residue Inhibition

The culture after 48 hours was compared with that before fermentationand evaluated.

Viable Cell Maintenance

The culture after 48 hours was suspended. The suspension was suitablydiluted, spread-plated onto MRS agar medium, and evaluated.

Melanogenesis Inhibition

(i) 20 ml of PBS and 0.2 ml of polyoxyethylene octylphenyl ether (TritonX-100) were mixed and stirred.

(ii) 10 ml of PBS and 19.7 ml of L-DOPA were mixed, and the mixture waswrapped in aluminum foil and stirred.

(iii) 90 μl of (i) and 10 μl of (ii) were added to the fermented extractand B16 melanoma cells (3000 cells/well) cultured for 2 days, and lightabsorbance (at 490 nm) at 0 and 60 minutes was measured with amicroplate reader and evaluated.

Oxidation Inhibition

The color of the medium 5 days after the melanin production inhibitiontest was confirmed with the naked eye, and oxidation of DOPA wasevaluated.

TABLE 4 Melanin pH Residue Viable cell production Oxidation reductioninhibition maintenance inhibition inhibition Example 1 + −/+ + + ++Example 2 + ++ +++ +++ +Production Example of Lactic Acid Bacteria Fermented Extract Lotion

1,000 mL of a solution containing 0.1% sodium citrate, 1.0% sodiumpyrrolidone carboxylate, and 5.0% 1,3-butylene glycol was prepared bymixing each component into purified water at 50° C. 100 mL of ethanolcontaining 0.6% POE(30)POP(6) decyltetradecyl ether (NIKKOL PEN-4630)was gradually added with stirring to the solution and therebysolubilized. The resulting product was cooled to 30° C. with stirringand the lactic acid bacteria fermented extract obtained in Example 2 wasadded to a final concentration of 0.5%, thus providing 1,000 mL of alactic acid bacteria fermented extract lotion.

What is claimed is:
 1. A method for: (a) overcoming or reducing risk ofinfection by a pathogen or virus that invades through the respiratory oresophageal mucosa; (b) alleviating or reducing risk of food poisoning;(c) regulating intestinal function; or (d) beautification; comprisingadministering to a subject in need thereof Lactobacillus kunkeei BPS402(FERM BP-11439) or treated cells thereof, or Lactobacillus kunkeeiBPS104 (FERM BP-11438) or treated cells thereof, wherein theadministering is effective for overcoming or reducing risk of infectionby the pathogen or virus that invades through the respiratory oresophageal mucosa, for alleviating or reducing risk of food poisoning,for regulating intestinal function, or for beautification.
 2. The methodof claim 1, wherein the administering is effective for overcoming orreducing risk of infection by the pathogen or virus that invades throughthe respiratory or esophageal mucosa.
 3. The method of claim 1, whereinthe administering is effective for alleviating or reducing risk of foodpoisoning.
 4. The method of claim 1, wherein the administering iseffective for regulating intestinal function.
 5. The method of claim 1,wherein the administering is effective for beautification or anti aging.6. The method of claim 1, wherein the administering is oral.
 7. Themethod of claim 1, wherein the Lactobacillus kunkeei BPS402 (FERMBP-11439) or treated cells thereof, or Lactobacillus kunkeei BPS104(FERM BP-11438) or treated cells thereof, are administered in granules.